Bolting, defined as stem elongation, marks the plant life cycle transition from vegetative to reproductive stage. Lettuce is grown for its leaf rosettes, and premature bolting may reduce crop quality resulting in economic losses. The transition to reproductive stage is a complex process that involves many genetic and environmental factors. In this study the effects of photoperiod and ambient temperature on bolting and flowering regulation was studied by utilizing a lettuce mapping population to identify quantitative trait loci (QTL) and by gene expression analyses of genotypes with contrasting phenotypes. A recombinant inbred line (RIL) population, derived from a cross between PI251246 (early bolting) and cv. Salinas (late bolting), was grown in four combinations of short (8 hours) and long (16 hours) days and low (20°C) and high (35°C) temperature. QTL models revealed both genetic (G) and environmental (E) effects, and GxE interactions. A major QTL for bolting and flowering time was found on chromosome 7 (qFLT7.2) and two candidate genes were identified by fine mapping, homology and gene expression studies. In short days and high temperature conditions qFLT7.2 had no effect on plant development, while several small-effect loci on chromosomes 2, 3, 6, 8 and 9 were associated with bolting and flowering. Of these, the QTL on chromosome 2, qBFr2.1, co-located with the Flowering Locus T (LsFT) gene. Polymorphisms between parent genotypes in the promotor region may explain identified gene expression differences, and were used to design a genetic marker which may be used to identify the late bolting trait.